Two common nonsynonymous paraoxonase 1 (<Emphasis Type="Italic">PON1</Emphasis>) gene polymorphisms and brain astrocytoma and meningioma

Background  

Human serum paraoxonase 1 (PON1) plays a major role in the metabolism of several organophosphorus compounds. The enzyme is encoded by the polymorphic gene PON1, located on chromosome 7q21.3. Aiming to identify genetic variations related to the risk of developing brain tumors, we investigated the putative association between common nonsynonymous PON1 polymorphisms and the risk of developing astrocytoma and meningioma.     

Occurrence and development of Eucryptorrhynchus brandti and E. chinensis (Coleoptera: Curculionidae) on Ailanthus altissima trees subjected to different levels of mechanical damage

Eucryptorrhynchus brandti (Harold) and E. chinensis (Oliver) are herbivores of Ailanthus altissima (Mill.) Swingle (tree of heaven) in China. Eucryptorrhynchus brandti has been recommended as a potential biological control agent of A. altissima in North America. In China, the majority of adults of both species were found on tree trunks 0–5 m above the ground, from May to September. In October and November, most adults were found at the base of A. altissima trees. Trees were subjected to different levels of mechanical injury: very low mechanical injury, one to two 0.015 m² bark sections were removed; low, two to four bark sections were removed; moderate, 8 to 22 bark sections removed; high, tree was girdled; and extreme, tree was felled and the remaining stump was sampled. Very low and low mechanical injury trees had very few adult emergence holes with few larvae developing beyond the first or second instar after 29 months. The health of these trees remained good. An increase in adult emergence holes occurred in moderately injured trees that appeared as healthy as very low to low mechanically injured trees. This may indicate that only a small increase in tree stress can increase the adult emergence rate and thus hasten a decline in tree health. Moderate, high and extreme mechanically injured trees had significantly more emergence holes/m than very low and low injured trees and late larval stages were present. The development of Eucryptorrhynchus species was more successful the greater the degree of mechanical injury. Based on this study both Eucryptorrhynchus species appear to be secondary pests of A. altissima in China.     

Survey of Verticillium nonalfalfae (Phyllachorales) on tree-of-heaven in the southeastern USA

Ailanthus altissima (Mill.) Swingle commonly known as tree-of-heaven is an invasive tree species that has spread through much of the USA. Significant mortality of A. altissima by a highly host-specific strain of the fungus, Verticillium nonalfalfae Inderb. (formerly Verticillium albo-atrum Reinke and Bethold), has been recently documented in Pennsylvania. To detect the regional distribution of this biological control agent in the Southeast, a windshield survey was conducted in Virginia, North Carolina and South Carolina to locate V. nonalfalfae-infected A. altissima stands. The survey covered 26,500 km of interstate highways and primary and secondary roads. There were 90 potential sites located throughout the three states. Of those, six were found to be infested with the fungus, all in the mountain regions of Virginia. Inoculation tests suggest no difference in virulence among the six V. nonalfalfae isolates on A. altissima, which resulted in 100% mortality in 9–11 weeks post-inoculation. Tree ratings revealed differences in health among sites, but all sites had an increase in disease incidence from 2011 to 2012 (mean = 4.7 ± 3.8%), suggesting this fungus is dispersing. This survey provides evidence that biological control using this fungus may be possible in Virginia.     

Use of morphometric parameters for tracking ovule and microspore evolution in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L., cv. “Aragonez”) and evaluation of their potential to improve <Emphasis Type="Italic">in vitro</Emphasis> somatic embryogenesis efficiency from gametophyte tissues

Somatic embryogenesis induction from in vitro cultured stamens and carpels is highly dependent on explants’ inoculation at specific developmental stages. To establish good correlations between measurable morphometric parameters of flowers or flower buds and developmental stages of micro- and macrosporogenesis, this procedure is the easiest way to simplify the in vitro culture procedures. These correlations were established here for the most important Iberian grapevine cultivar, the “Aragonez”, named “Tempranillo” in Spain and “Tinta Roriz” in the north of Portugal, and were based in floral buds and anther measurements. The anther length, with a correlation coefficient of 0.90, proved to be the best morphometric parameter to follow microsporogenesis evolution. A correlation between micro- and macrosporogenesis evolutionary stages was also positively established, allowing the use of morphometric parameters for tracking ovule evolution as well. Carpels in several evolutionary stages were in vitro cultured to evaluate the aging effect on the capacity for somatic embryogenesis induction. Explants inoculated in the earliest stages of macrosporogenesis presented the best results. Media culture formulations were also tested for ovary culture, with the best results being achieved with a 5:1 auxin/cytokinin ratio.     

High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion

Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA-protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.     

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.     

Confined diffusion without fences of a g-protein-coupled receptor as revealed by single particle tracking

Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.